Daniel:Notebook/ComboLock/2017-3-26
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RCA Test
Linear Product Production-Bglii
- TBE gel
- Mix 64 uL TBE and 16 uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot File:GelLanes-20170325-LinearProduct.png
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
File:2017-03-26-LinearProductProduction-Bglii.png
That's the right one. However, there is a ton of primer (bottom of the gel), so I'll run a ssDNA column to fix.
- ssDNA column
- Add 50 uL Binding Buffer to the sample; mix well
- Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
- Add 70 uL 100% EtOH to flow through; mix well
- Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Centrifuge empty column at 14000 rpm for 2 minutes
- Transfer to empty 1.5mL centrifuge tube (low bind)
- Add 20 uL nfH2O and centrifuge at 14000 rpm for 1 minute